cDNA synthesis
Usually after we have extracted RNA from tissue we will synthesize
cDNA from 500ug DNase treated RNA. We use the Promega kit to
synthesize cDNA in a total reaction volume of 20ul. I have detailed
our protocol below.
During the synthesis of cDNA we also setup 2 negative controls, a
very important step in the process. Our negative controls are prepared
with the same mix as is used with our study group and consist of:
1) One negative consisting of a duplicate of one sample (selected at
random) but without the RT enzyme added.
2) One negative consisting of the RT enzyme and synthesis mix but
with no RNA and just nuclease free water.
cDNA synthesis Protocol:
1) 500ng RNA of each sample prepared in volume of 10ul and placed
on ice.
2) 1 negative control prepared without RNA in 10ul water (labelled
RNA-VE)
3) 1 negative control containing 500ng RNA from any one sample
(labelled RT-VE)
4) If you wish, make extra control sample to use for the Standard
curve and label SC (Alternatively you can use a pool of your control
cDNA for this).
5) Prepare mix:
(1x)
5x Mastermix
4ul
MgCl2 (25mM)
2ul
Random Hexamers (100ug/ml) 0.5ul
dNTP mix (10mM)
2.5ul
9ul
6) 9ul of mix added to each sample and 2 negative controls
7) All samples heated to 65C for 5 minutes and returned to ice
8) 1ul RT enzyme added to all samples (NOT added to RT-VE)
9) All samples heated at 37C for 60 minutes.
10) Samples frozen or 1:30 dilution made if running 96 well plates in
which case SC, 1:20 dilution made.
11) For 384 well plate follow other protocol.